Compositions and methods for the inhibition of neurotransmitter uptake of synaptic vesicles

ABSTRACT

Compositions and methods for treating neurosynaptic disorder in a subject are described. More specifically, compositions and methods for inhibiting glutamate uptake by synaptic vesicles in a subject are set forth. In one embodiment, the composition is inhibitory protein factor (IPF) and the subject is a human.

This is a Divisional of application Ser. No. 08/840,006 filed on Apr.15, 1997, now U.S. Pat. No. 6,127,520.

This invention was made with government support under JavitsNeuroscience Investigator Award NS 26884 awarded by the NationalInstitutes of Health, Department of Health and Human Services. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention pertains to compositions and methods for theinhibition of neurotransmitter uptake of synaptic vesicles.

BACKGROUND

Glutamate is now widely accepted as the major excitatoryneurotransmitter in the central nervous system of all vertebrates [seee.g., Nakanishi (1992) Science 258, 597–603]. Abnormalities inglutamatergic synaptic transmission have been implicated in manyneuropathologies, including certain seizures, ischemia-induced neuronaldeath, schizophrenia, Alzheimer's disease, Parkinson's disease, andHuntington's disease [see e.g., Coyle and Puttfarcken (1993) Science262, 689–695].

Excessive release of glutamate into the synaptic cleft is believed to bea common underlying basis for many of these disease states. There isalso evidence that some glutamate receptors such as the NMDA(N-methyl-D-aspartate) and the metabotropic receptors may be involved inneuronal plasticity [see e.g., Bashir et al. (1993) Nature 363,347–350].

Evidence which has been accumulated for the last decade stronglysupports the notion that glutamatergic neurotransmission occurs via anexocytotic process involving the interaction of glutamate-containingsynaptic vesicles with the plasma membrane of the presynaptic ending. Insupport of this is the observation that glutamate is taken up intopurified, isolated synaptic vesicles in an ATP-dependent manner [seee.g., Naito and Ueda (1983) J. Biol. Chem. 258, 696–699; and Tabb andUeda (1991) J. Neurosci. 11, 1822–1828], consistent withimmunocytochemical evidence that glutamate is concentrated in synapticvesicles which are distinct from GABA-vesicles [Storm-Mathisen et al.(1983) Nature 301, 517–520].

Biochemical evidence also suggested that high concentrations ofglutamate are accumulated in brain synaptic vesicles in vivo. Studies byNicholls and co-workers indicate that the exocytotic pool of glutamateoriginates from a noncytoplasmic site within the nerve terminal [seee.g., Nicholls and Sihra (1986) Nature 321, 772–773; and McMahon andNicholls (1991) Biochim. Biophys. Acta 1059, 243–264].

Moreover, Kish and Ueda [(1991) Neurosci. Lett. 122, 179–182] providedevidence that vesicular glutamate is released in a calcium-dependentmanner from permeabilized synaptosomes. This body of evidence clearlydemonstrates that synaptic vesicles are the storage site of theglutamate to be released from nerve terminals.

The vesicular glutamate uptake system has several distinctive propertieswhich distinguish it from the cellular glutamate re-uptake systempresent in the plasma membrane. It is stringently specific forglutamate, has a relatively high K_(m), and requires low concentrationsof chloride for optimal activity [(Naito and Ueda (1985) J. Neurochem.44, 99–109; and Fykse et al. (1989) J. Neurochem. 52, 946–951].

The driving force for glutamate uptake is provided by an electrochemicalproton gradient generated by a V-type H⁺-ATPase in the synaptic vesiclemembrane [Naito and Ueda (1985) J. Neurochem. 44, 99–109]. The precisemechanism by which the glutamate transporter utilizes this protongradient to drive glutamate uptake is not fully understood; however,compounds that interfere with the formation of such gradients have amarked inhibitory effect on glutamate transport [Naito and Ueda (1985)J. Neurochem. 44, 99–109; Tabb et al. (1992) J. Biol Chem 267,15412–15418].

It has been proposed that glutamate uptake into synaptic vesiclesrepresents the critical step in diverting glutamate away from themetabolic pathway and toward the neurotransmitter pathway [Ueda (1986)in Excitatory Amino Acids (Roberts P. J., Storm-Mathisen J., andBradford H. F., eds), pp. 173–195, Macmillan Press, London]. Thus, it isdesirable to regulate the vesicular glutamate uptake system under normalphysiological conditions. Alterations in such a regulatory system couldcause the abnormalities in glutamatergic neurotransmission implicated inthe variety of central nervous system disorders mentioned above.However, while many studies have focused on changes associated withpostsynaptic glutamate receptors, few have addressed presynapticregulation of glutamatergic neurotransmission at the level of vesiculartransport. What is needed are compositions and methods for regulatingthe uptake of glutamate into the synaptic vesicles.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for the inhibition ofneurotransmitter uptake of synaptic vesicles. In one embodiment, thepresent invention provides compositions and methods for regulating theuptake of glutamate by synaptic vesicles. In one embodiment, the presentinvention contemplates the inhibition of glutamate uptake by synapticvesicles with an inhibitor. In one embodiment, the inhibitor is apeptide, and in a preferred embodiment, the peptide is inhibitoryprotein factor (IPF).

The term “IPF” is used in reference to three proteins (IPF αβγ) eachhaving an apparent molecular weight on electrophoresis gels of between130 and 138 kD. IPF can be purified from animal brain tissue, includinghuman tissue.

In one embodiment, the present invention contemplates a compositioncomprising a purified fragment of fodrin having glutamate uptakeinhibition activity, said fragment having an N-terminus and aC-terminus. The composition is not limited by the exact amino acidsequence, however in one embodiment the N-terminus of the fragmentcorresponds to Tyr²⁶ of fodrin (that is to say, the N-terminus of thefragment begins with this amino acid and has additional amino acids thatfollow after this initial amino acid in the sequence order found inintact fodrin—although it is not intended to be limited to any preciselength). In another embodiment, the purified fragment comprises apeptide having the amino acid sequence (SEQ ID NO:1) EAALTSEEVG within150 amino acids of the C-terminus of the peptide, more preferably,within 130 amino acids of the C-terminus of the peptide, and mostpreferably, within 120 amino acids of the C-terminus of the peptide.

Likewise, the present invention is not limited by the precise size ofthe fragment of fodrin. In one embodiment, the purified fragmentcomprises IPF α, while in other embodiments the composition comprises apurified fragment of IPF α. In yet other embodiments, the purifiedfragment comprises IPF β or IPF γ.

While the present invention is not limited to a specific amino acidsequence, in one embodiment the present invention contemplates acomposition comprising a purified peptide having glutamate uptakeinhibition activity with an N-terminus sequence comprising the aminoacids (SEQ ID NO:2) YHRFK (i.e., the peptide N-terminus begins with thissequence and has at least these amino acids in this order). In anotherembodiment, the said purified peptide has an N-terminus comprising theamino acids (SEQ ID NO:3) YHRFKELSTL (i.e., the peptide N-terminusbegins with this sequence and has at least these amino acids in thisorder). In yet another embodiment, the purified peptide has anN-terminus comprising the amino acids (SEQ ID NO:4) YHRFKELSTLRRQKLEDSYR(i.e., the peptide N-terminus begins with this sequence and has at leastthese amino acids in this order).

The present invention also contemplates a method of isolating aglutamate uptake inhibitor, comprising: a) providing an animal brain;and b) subjecting said animal brain to a purification procedure suchthat a purified fodrin fragment having glutamate uptake inhibitionactivity is produced. The present invention is not limited by thespecific purified fragment. In certain embodiments the purified fragmentcomprises IPF α, IPF β or IPF γ.

The present invention contemplates screening assays for candidatecompounds. While the present invention is not limited by any particularscreening assay, in one embodiment, the present invention contemplates amethod for testing compounds for their ability to overcome or offset thesynaptic vesicle glutamate uptake inhibition activity of the fodrinfragments of the present invention. In one embodiment, the screeningmethod comprises a) providing: i) synaptic vesicles, ii) as purifiedfodrin fragment having glutamate uptake inhibition activity, and iii)candidate compound; and b) combining said candidate compound with saidsynaptic vesicles and said fragment such that the effect of saidcandidate compound on glutamate uptake by said synaptic vesicles can beassessed. The method is not limited by the purified fragment utilized,in certain embodiments the purified fragment comprises IPF α, IPF β orIPF γ.

Another screening assay contemplated by the present inventioncontemplates a method for testing compounds for their ability toovercome or offset the synaptic vesicle glutamate uptake inhibitionactivity of fragments of IPF. In one embodiment, the method comprises a)providing: i) synaptic vesicles, ii) a purified fragment of IPF havingsynaptic vesicle glutamate uptake inhibition activity, and iii)candidate compound; and b) combining said candidate compound with saidsynaptic vesicles and said purified fragment such that the effect ofsaid candidate compound on said fragment's effect on glutamate uptake bysaid synaptic vesicles can be assessed. The assay is not limited by thenature of the purified fragments, however in certain embodiments thepurified fragment comprises fragments of IPF α, IPF β or IPF γ.

The present invention also contemplates screening method of testingcandidate compound for inhibition of calpain cleavage of fodrin,comprising: a) providing: i) fodrin, ii) calpain, and iii) candidatecompound; and b) combining said candidate compound with said fodrin andsaid calpain such that the effect of said candidate compound on calpaincleavage of fodrin can be assessed. In one embodiment, antibodies tofodrin are used as control inhibitors of calpain digestion.

In yet another embodiment, the present invention contemplates ascreening method of testing candidate compound for inhibition of trypsincleavage of IPF, comprising: a) providing: i) trypsin, ii) purified IPF,and iii) candidate compound; and b) combining said candidate compoundwith said trypsin and said IPF such that the effect of said candidatecompound on cleavage of said IPF by said trypsin can be assessed. In oneembodiment, antibodies to IPF a fragments are used as control inhibitorsof trypsin cleavage. The screening method is not limited by the natureof the purified IPF utilized. In certain embodiments, the purifiedfragment comprises IPF α, IPF β or IPF γ.

The present invention also contemplates antibodies to fodrin andpurified fragments of fodrin. While the present invention is not limitedby the sequence of the epitope of the purified fragment, in oneembodiment the epitope sequence corresponds to a portion of adecapeptide from fodrin. In yet another embodiment the antibodies havebeen passed through a column containing purified fodrin. In stillanother embodiment, the antibodies will bind to a purified fragment offodrin, but do not bind to fodrin.

The present invention also contemplates immobilized fodrin andfragments, such as fragments bound to a resin. The present invention isnot limited by the specific nature of the purified fragment bound. Incertain embodiments the purified fragment comprises IPF α, IPF β or IPFγ. In yet another embodiment, the purified fragment comprises a fragmentof IPF α.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a series of halogenated glutamic acid analogs useful forinhibiting synaptic vesicle glutamate uptake.

FIG. 2 depicts the SDS-PAGE profiles of fractions obtained during thepurification of IPF.

FIG. 3 compares the SDS-PAGE profile (lower panel) of peak inhibitoryfractions as eluted from Mono Q HPLC (upper panel).

FIG. 4 depicts a dose-response curve for purified IPF α. As theconcentration of IPF is increased, the ATP-dependent glutamate uptake isdecreased.

FIG. 5 graphically depicts the specificity of the inhibitory effectproduced by IPF α.

FIG. 6A compares partial IPF α sequence (SEQ ID NO:5) with the sequenceof human α fodrin, while FIG. 6B shows the predicted sequence (SEQ IDNO:6) of IPF α based upon the C-terminus being commensurate with thecalpain cleavage site for fodrin and the N-terminus beginning at aminoacid 26 of fodrin.

FIG. 7 shows the effect of trypsin exposure on IPF. Lane A shows theSDS-PAGE profile of the partially purified IPF composition withouttrypsin treatment. Lane B shows the SDS-PAGE profile of the partiallypurified IPF composition with trypsin treatment. It is clear that the138 kD IPF band is not present, demonstrating the digestion of IPF bytrypsin. Lane C shows the SDS-PAGE profile of the partially purified IPFcomposition in the presence of trypsin and pancreatic trypsin inhibitor.

FIG. 8 depicts the results of the fractions obtained from FIG. 7. Lane Adepicts control glutamate uptake without additives. Lane B depictsglutamate uptake by synaptic vesicles in the presence of trypsin andpancreatic trypsin inhibitor. Lane C depicts glutamate uptake bysynaptic vesicles in the presence of partially purified IPF. Lane Ddepicts glutamate uptake by synaptic vesicles in the presence ofpartially purified IPF and trypsin. Lane E depicts glutamate uptake bysynaptic vesicles in the presence of partially purified IPF, trypsin andpancreatic trypsin inhibitor.

FIG. 9 depicts the results of Western Blot assays of antibodies raisedto a fodrin decapeptide. Panel A demonstrates the binding of antibodiesto both fodrin and IPF, while Panel B demonstrates binding of antibodiesto IPF without binding to fodrin.

DEFINITIONS

“Glutamate uptake inhibition activity” as used herein refers to theproperty of inhibiting the uptake of glutamate by synaptic vesicles. A“glutamate uptake inhibitor” is a peptide that demonstrates glutamateuptake inhibition activity.

“IPF” as used herein refers to a peptide derived from animal braintissue that is a glutamate uptake inhibitor. IPF is a designation forthree similar peptides “IPF αβγ.”

“Neurosynaptic disorder” as used herein refers to undesirable neuronalactivity resulting in seizures of a subject or damage to a subject'sneural tissue. Examples of such conditions include, but are not limitedto, epileptic seizures, Huntington's Disease, Alzheimer's Disease, etc.

“GABA” as used herein refers to γ-aminobutyric acid and acts as aninhibitory transmitter in the central nervous system.

“Fodrin” as used herein refers to a protein of a family of proteins thatbundle and crosslink actin filaments. Actin filaments are a part of thecytoskeleton and are responsible for maintaining cell structure andintegrity. Fodrin is a rod-shaped protein that lines the corticalcytoplasm of neurons. Fodrin can be identified as a high molecularweight protein present in brain membranes by i) comigration on NaDodSO₄polyacrylamide gels with purified fodrin, ii) reactivity with antibodiesto purified fodrin, and iii) a proteolytic map following calpainactivation comparable to that found after calpain mediated degradationof pure fodrin. “α fodrin” as used herein refers to the fodrin asisolated from human cells. “Fodrin fragment” as used herein refers to apeptide or protein whose amino acid sequence is equivalent to a portionof the amino acid sequence of fodrin. “Purified fodrin fragment” as usedherein refers to a fodrin fragment that is isolated from the naturalenvironment of fodrin or a fodrin fragment created by degradation offodrin that has been isolated from its natural environment.

“Calpain” as used herein refers to a calcium calmodulin dependentneutral proteinase isolated from the cytostolic fractions of variousanimal tissues or cells with a molecular weight of 94–100 kDa by gelfiltration on Sephacryl 300.

“Trypsin” as used herein refers to a proteolytic enzyme from pancreaticjuice that hydrolyses polypeptides on the carboxyl side of arginine andlysine residues.

“Trypsin sensitive” as used herein refers to a peptide with an arginineor lysine residue that is sensitive to cleavage by trypsin on thecarboxyl side.

“Purified” as used herein refers to a composition wherein at least onecomponent has been removed from the crude extract causing the proportionof protein of interest (e.g., IPF or fodrin) to be increased relative tothe total proteins found in a crude extract.

“Apparent molecular weight” as used herein refers to the estimatedmolecular weight as determined by methods (e.g., gel electrophoresis,sucrose gradient, gel filtration) and compared to the molecular weightsof standards run by the same method.

“True molecular weight” as used herein refers to the molecular weight ofa peptide as determined by the additive molecular weights of itscomponent amino acids as determined by complete sequencing of thepeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to compositions and methods for theinhibition of neurotransmitter uptake of synaptic vesicles. In apreferred embodiment, the inhibitor is IPF. In another embodiment, theinhibitor is a product of exposure of IPF to trypsin.

IPF is isolated from brain tissue and refers to three distinct proteinswith relative molecular weights of 138,000 (α), 135,000 (β), and 132,000(γ), respectively. It has been determined that IPF is best purified frombrain material excised from young animals as opposed to mature animals.

While an understanding of the precise mechanism is not necessary tocarry out the present invention, it is believed that IPF interacts witha synaptic vesicle-specific protein that leads to a blockade ofneurotransmitter storage by an indirect mechanism. The physiologicalrole of IPF remains to be elucidated. The fact that IPF seems to derivefrom α fodrin is particularly intriguing since fodrin purified fromwhole brain is itself devoid of inhibitory activity (data not shown).Regardless of the mechanism of action of IPF, it is a potent, endogenousinhibitor of vesicular neurotransmitter uptake.

The present invention also contemplates degradation products of IPF thatexhibit synaptic vesicle glutamate uptake inhibition and the usethereof. While the present invention is not limited by the type ormethod of producing these degradation products, in one embodiment IPF isdegraded with trypsin and these degradation products inhibit glutamateuptake by synaptic vesicles.

Mimetics

Compounds mimicking the necessary conformation for recognition anddocking to the receptor binding to the peptides of the present inventionare contemplated as within the scope of this invention. For example,mimetics of IPF peptides are contemplated. A variety of designs for suchmimetics are possible. For example, cyclic IPF-containing peptides, inwhich the necessary conformation for binding is stabilized bynonpeptides, are specifically contemplated. U.S. Pat. No. 5,192,746 toLobl et al, U.S. Pat. No. 5,169,862 to Burke, Jr., et al, U.S. Pat. No.5,539,085 to Bischoff et al, U.S. Pat. No. 5,576,423 to Aversa et al,U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat. No. 5,559,103 toGaeta et al, all hereby incorporated by reference, describe multiplemethods for creating such compounds.

Synthesis of nonpeptide compounds that mimic peptide sequences is alsoknown in the art. Eldred et al, (J. Med. Chem. 37:3882 (1994)) describenonpeptide antagonists that mimic the Arg-Gly-Asp sequence. Likewise, Kuet al, (J. Med. Chem. 38:9 (1995)) give further elucidation of thesynthesis of a series of such compounds. Such nonpeptide compounds thatmimic IPF peptides are specifically contemplated by the presentinvention.

The present invention also contemplates synthetic mimicking compoundsthat are multimeric compounds that repeat the relevant peptide sequence.As is known in the art, peptides can be synthesized by linking an aminogroup to a carboxyl group that has been activated by reaction with acoupling agent, such as dicyclohexylcarbodiimide (DCC). The attack of afree amino group on the activated carboxyl leads to the formation of apeptide bond and the release of dicyclohexylurea. It can be necessary toprotect potentially reactive groups other than the amino and carboxylgroups intended to react. For example, the α-amino group of thecomponent containing the activated carboxyl group can be blocked with atertbutyloxycarbonyl group. This protecting group can be subsequentlyremoved by exposing the peptide to dilute acid, which leaves peptidebonds intact.

With this method, peptides can be readily synthesized by a solid phasemethod by adding amino acids stepwise to a growing peptide chain that islinked to an insoluble matrix, such as polystyrene beads. Thecarboxyl-terminal amino acid (with an amino protecting group) of thedesired peptide sequence is first anchored to the polystyrene beads. Theprotecting group of the amino acid is then removed. The next amino acid(with the protecting group) is added with the coupling agent. This isfollowed by a washing cycle. The cycle is repeated as necessary.

In one embodiment, the mimetics of the present invention are peptideshaving sequence homology to the above-described IPF peptides. One commonmethodology for evaluating sequence homology, and more importantlystatistically significant similarities, is to use a Monte Carlo analysisusing an algorithm written by Lipman and Pearson to obtain a Z value.According to this analysis, a Z value greater than 6 indicates probablesignificance, and a Z value greater than 10 is considered to bestatistically significant. W. R. Pearson and D. J. Lipman, Proc. Natl.Acad. Sci. (USA), 85:2444–2448 (1988); D. J. Lipman and W. R. Pearson,Science, 227:1435–1441 (1985). In the present invention, syntheticpolypeptides useful in glutamate uptake inhibition by synaptic vesiclesare those peptides with statistically significant sequence homology andsimilarity (Z value of Lipman and Pearson algorithm in Monte Carloanalysis exceeding 6).

Glutamate Analogs as Inhibitors of Glutamate Uptake in Synaptic Vesicles

The present invention contemplates the use of glutamate analogs toinhibit glutamate uptake by synaptic vesicles. While not being limitedby the nature of the glutamate analogs, halogenated analogs of glutamicacid and cyclic analogs of glutamate are contemplated.

While the present invention is not limited by the type of halogenatedglutamic acid analog utilized, examples of such compounds are set forthin FIG. 1. Where the structures set forth signify an X substituent, anyof the halogens (i.e., fluorine, chlorine, bromine or iodine) may beused.

For cyclic analogs of glutamate, it is contemplated that compounds thathave a three-dimensional charge distribution identical or very close tothat of (1S,3R)-1-aminocylcopentane-1,3-dicarboxylate (ACPD), an analogof L-glutamate, are useful. Preferably, the compound has a hydrophobicmoiety similar to that seen in (1S,3R)-ACPD. Examples include, but arenot limited to, 1-aminocylcobutane-1,3-dicarboxylic acid,1-aminocylcohexane-1,3-dicarboxylic acid and1-aminocylcoheptane-1,3-dicarboxylic acid [all available commerciallyfrom Tocris-Cookson, Bristol, UK]. The present invention is not limitedby the form of the cyclic analog of glutamate. For example, derivativesof cyclic analogs (e.g., halogenated, methylated or ethylated) are alsocontemplated.

Drug Screening Assays

The present invention contemplates in vitro screening assays for thediscovery of 1) new glutamate uptake inhibitors, 2) compounds that canovercome glutamate uptake inhibition, 3) inhibitors of calpaindegradation of fodrin, 4) inhibitors of trypsin degradation of IPF and5) compounds that can overcome glutamate uptake inhibitor by the trypsindegradation products of IPF. The present invention also contemplates invivo screening assays to assess efficacy.

These screening assays are described in detail below. Glutamate andsynaptic vesicles are utilized in the in vitro assays. The presentinvention is not limited by the nature of the synaptic vesiclesutilized. For example, synaptic vesicles from bovine and mice [as setforth in Examples below] and from rats [Carlson et al. (1989) J.Neurochemistry 53 1889–1894], as well as other sources are contemplated.

1. New Glutamate Uptake Inhibitors

The present invention contemplates the use of IPF with glutamate andsynaptic vesicles to assess glutamate uptake inhibition of othercandidate inhibitors. While the present invention is not limited by theactual assay protocol, IPF can be used as a standard for testing theinhibition properties of other candidate compounds.

In such an assay, glutamate uptake inhibition by synaptic vesicles isassessed as set forth in the Examples below. Such uptake can be measuredwith and without the presence of IPF. Meanwhile, candidate inhibitor canbe assayed under similar conditions in the absence of IPF. The extent ofglutamate uptake by the synaptic vesicles in the presence of candidateinhibitor can then be compared to uptake with no additive and uptake inthe presence of IPF. If uptake in the presence of candidate inhibitorreduces glutamate uptake by synaptic vesicles as compared to uptake inthe absence of candidate inhibitor, the candidate inhibitor isconsidered to exhibit glutamate uptake inhibition activity.

2. Compounds That Overcome Glutamate Uptake Inhibition

The present invention contemplates the screening of compounds useful forovercoming inhibition of glutamate uptake by synaptic vesicles. Whilethe present invention is not limited by the actual assay protocol, it iscontemplated that candidate compounds can be screened for their abilityto overcome IPF inhibition of glutamate uptake by synaptic vesicles.

In such an assay, glutamate uptake by synaptic vesicles is inhibited byIPF as set forth in the Examples below. Candidate compound can be addedand uptake by synaptic vesicles can be assessed. If glutamate uptake bysynaptic vesicles in the presence of IPF and candidate compound isincreased in relation to uptake in the presence of IPF alone, thecandidate compound overcomes glutamate uptake inhibition activity bysynaptic vesicle glutamate uptake inhibitors.

3. Inhibitors of Calpain Degradation of Fodrin

It is believed that the C-terminus of IPF corresponds with a calpaincleavage site of fodrin. It is therefore hypothesized that theC-terminus region of IPF can be formed from fodrin by cleavage withcalpain. This cleavage should result in a fragment of fodrin of about150 kD, with a C-terminus matching that of IPF and an N-terminus similarto that of fodrin. This fragment is in contrast to IPF itself of about138 kD, whose N-terminus corresponds with the amino acid sequence offodrin beginning at position 26 (i.e., lacking the first 25 amino acidsof fodrin).

The present invention contemplates the screening of compounds useful forinhibiting calpain cleavage of fodrin. While the present invention isnot limited by the actual assay protocol, it is contemplated thatcandidate compounds can be screened for their ability to inhibit calpaincleavage of fodrin by introducing candidate compound with fodrin andcalpain, and assessing the presence and nature of any cleavage products(e.g., by examining the products on gel electrophoresis).

In such an assay, if the cleavage products in the presence of candidatecompound do not correspond with the 150 kD or 138 kD proteins describedabove, the candidate compounds is useful as a inhibitor of fodrincleavage by calpain.

4. Inhibitors of Trypsin Degradation of IPF

As set forth in the Examples below, it has been determined that trypsincan cleave IPF. These degradation products have synaptic vesicleglutamate uptake inhibition activity similar to that of IPF itself.

The present invention contemplates the screening of compounds useful forinhibiting trypsin degradation of IPF. While the present invention isnot limited by the actual assay protocol, it is contemplated thatcandidate compounds can be screened for their ability to inhibit trypsincleavage of IPF by introducing candidate compound with IPF and trypsin,and assessing the presence and nature of any cleavage products.

In such an assay, if the cleavage products in the presence of candidatecompound do not correspond with the trypsin cleavage products describedabove, the candidate compounds is useful as a inhibitor of IPF cleavageby trypsin.

5. Overcoming Glutamate Uptake Inhibition by IPF Fragments

As described above, fragments of IPF exhibit synaptic vesicle glutamateuptake inhibition activity. While the present invention is not limitedby the nature of the fragments, the present invention does contemplatethe screening of compounds useful for overcoming such IPF fragmentinhibition of glutamate uptake by synaptic vesicles.

In such an assay, glutamate uptake by synaptic vesicles is inhibited byIPF fragments as set forth in the Examples below. Candidate compound canbe added and uptake by synaptic vesicles can be assessed. If glutamateuptake by synaptic vesicles in the presence of IPF fragments andcandidate compound is increased in relation to uptake in the presence ofIPF fragments alone, the candidate compound overcomes glutamate uptakeinhibition activity by IPF fragments.

6. In Vivo Assays of Glutamate Uptake Inhibitors

The present invention contemplates the treatment of synaptic disorder insubjects and is not limited by the type of glutamate uptake inhibitorutilized. Candidate inhibitors may be tested prior to administration toa patient. Once drugs possessing inhibitory activity and lackingneuronal toxicity are identified, they may be administered according tothe invention to a patient exhibiting symptoms of a disease or disorderassociated with neuronal injury or death, particularly to patients inwhich neuronal injury or death is a result of glutamate toxicity.Preferred inhibitors are those which are capable of crossing theblood/brain barrier.

The above-identified inhibitors and also drugs identified according tothe invention as inhibitors of glutamate uptake by synaptic vesicles canbe tested in animals for their effectiveness in inhibiting or preventingneuronal seizures and injury and/or death. Conventional animal testingsystems are well-known in the art. Each animal may be injected with arange of doses of the drug and the toxicity or lack of toxicity of thedrug may be assessed by the survival rate of the animals. Animals whichexhibit symptoms of neuronal injury, or animals subject to conditionsgenerating cerebral infarction (i.e., stroke), e.g, oxygen or glucosedeprived animals, can then be tested. The effects of the drug onperipheral tissues can be assessed by histological examination of thetissues. Based on these in vivo tests, the overall effectiveness of thedrug can be determined, and an effective dose and mode of administrationcan also be determined. The present invention is not limited by thenature of the animal or disease modeled. In one embodiment, the animalmodel is of a type as set forth in the Examples below (e.g., the ELmouse strain).

Drug Therapy and Formulations

The present invention is not limited by the method of administration ofthe inhibitor. In one embodiment, it is by conventional means availablefor use in conjunction with pharmaceuticals; either in combination withone another or in combination with other therapeutic agents. It iscontemplated that the methods of the present invention be administeredalone or can be administered with a pharmaceutical carrier selected onthe basis of the chosen route of administration and standardpharmaceutical practice. In one embodiment, the inhibitor isadministered by a implanting device for the release of neuroinhibitorycompounds. One example of such a device is described in U.S. Pat. No.5,573,528 to Aebischer et al., herein incorporated by reference.

In one preferred embodiment, the inhibitor is administered orally insolid dosage forms, such as capsules, tablets, or powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions; however, it canalso be administered parenterally, in sterile liquid dosage forms, orrectally in the form of suppositories.

One skilled in the art will be capable of adjusting the administereddose depending upon known factors such as the mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. In one embodiment, the dosage isincreased to overcome a non-responsive condition.

Additionally, the inhibitor can be employed in admixture withconventional excipient, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral (e.g., topicalapplication) or enteral (e.g., oral) which do not deleteriously reactwith the active compounds.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils,benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such aslactose, amylose, or starch, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, merely to name a few. Thepharmaceutical preparations can be sterilized and if desired mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifier, salts for influencing osmotic pressure, buffers,coloring, flavoring, and/or aromatic substances and the like which do nodeleteriously react with the active compounds. They can also be combinedwhere desired with other agents, e.g. vitamins.

Additionally, the inhibitor may be introduced in a polymer gel forcontrolled release. Methods of producing such gels are set forth in U.S.Pat. No. 5,567,435 to Hubbel et al., herein incorporated by reference.Methods of injecting polymers are set forth in U.S. Pat. No. 5,567,612to Vacanti et al., hereby incorporated by reference. Moreover, in caseswhere the inhibitor chosen is a peptide, methods for incorporatingpeptides into polymers is described in U.S. Pat. No. 5,482,996 toRussell et al., herein incorporated by reference.

For enteral application, particularly suitable are tablets, liquids,drops, suppositories, or capsules. A syrup, elixir, or the like can beused wherein a sweetened vehicle is employed. Sustained or directedrelease compositions can be formulated, e.g., liposomes or those whereinthe active compound is protected with differentially degradable coating,e.g., by microencapsulation, multiple coatings, etc.

In this manner, the present invention may be introduced into a subjectin polymeric microspheres for the controlled release of the compound.Methods of producing microspheres from polymer can be found in U.S. Pat.No. 5,601,844 to Kagayama et al. and U.S. Pat. Nos. 5,529,914 and5,573,934 to Hubbel et al., herein incorporated by reference.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants including suppositories. Nebulizersand inhalation aerosols may also be used. Ampules are in convenient unitdosages. It is also possible to freeze-dry the new compounds and use thelypophilizates obtained, for example, for the preparation of productsfor injection.

For other parenteral applications, such as topical applications andnon-sprayable forms, viscous to semi-solid or solid forms comprising acarrier compatible with topical application and having a dynamicviscosity preferably greater than water. Suitable formulations includebut are not limited to transdermal patches, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, aerosols,etc., which are, if desired, sterilized or mixed with auxiliary agents,e.g., preservations, stabilizers, wetting agents, buffers, or salts forinfluencing osmotic pressure, etc.

Also suitable for topical application are sprayable aerosol preparationswherein the inhibitor, preferably in combination with a solid or liquidinert carrier material, is packaged in a squeeze bottle or in admixturewith pressurized volatile, normally gaseous propellant, e.g., a freon.The application of these embodiments can be to the skin or mucousmembrane or in the interior of the body and can be oral, peroral,enteral, pulmonary, rectal, nasal, vaginal, lingual, intervenous,intraarterial, intracardial, intramuscular, intraperitoneal,intracutaneous, subcutaneous. The parenteral preparations are preferablysterile or sterilized products.

In this manner, U.S. Pat. No. 4,895,727 to Allen, herein incorporated byreference, describes a method of inducing a reservoir effect in skin andmucous membranes so as to enhance penetration and retention and reducetransdermal flux of topically applied therapeutic and cosmeticpharmacologically active agents. U.S. Pat. No. 4,557,934 to Cooper,herein incorporated by reference, describes topical pharmaceuticalcompositions containing a pharmaceutically-active agent and thepenetration enhancing agent, 1-dodecylazacycloheptan-2-one. Thiscomposition provides marked transepidermal and percutaneous delivery ofthe selected pharmaceutically-active agent.

Suppositories containing inhibitor can be created using a suitableoleaginous or water-soluble base. The oleaginous class includes cocoabutter and fats with similar properties: the water-soluble classincludes polyethylene glycols.

Other medicaments containing inhibitor can be produced in a knownmanner, whereby the known and customary pharmaceutical adjuvants as wellas other customary carrier and diluting agents can be used. Examplesinclude, but are not limited to, gelatins, natural sugars such assucrose or lactose, lecithin, pectin, starch (for example cornstarch),alginic acid, tylose, talc, lycopodium, silica (for example colloidalsilica), glucose, cellulose, cellulose derivatives for example,cellulose ethers in which the cellulose hydroxyl group are partiallyetherified with lower aliphatic alcohols and/or lower saturatedoxyalchohols, for example, methyl hydroxypropyl cellulose, methylcellulose, cellulose phthalate, stearates, e.g., methylstearate andglyceryl stearate, magnesium and calcium salts of fatty acids with 12 to22 carbon atoms, especially saturated acids (for example, calciumstearate, calcium laurate, magnesium oleate, calcium palmitate, calciumbehenate and magnesium stearate), emulsifiers, oils and fats, especiallyof plant origin (for example, peanut oil, castor oil, olive oil, sesameoil, cottonseed oil, corn oil, wheat germ oil, sunflower seed oil,cod-liver oil), mono, di, and triglycerides of saturated fatty acids(C₁₂H₂₄O₂ to C₁₈H₃₆O₂ and their mixtures), e.g. glyceryl monostearate,glyceryl distearate, glyceryl tristearate, glyceryl trilaurate),pharmaceutically compatible mono- or polyvalent alcohols and polyglycolssuch as glycerine, mannitol, sorbitol, pentaerythritol, ethyl alcohol,diethylene glycol, triethylene glycol, ethylene glycol, propyleneglycol, dipropylene glycol, polyethylene glycol 400, and otherpolyethylene glycols, as well as derivatives of such alcohols andpolyglycols, esters of saturated and unsaturated fatty acids (2 to 22carbon atoms, especially 10 to 18 carbon atoms), withmonohydricaliphatic alcohols (1 to 20 carbon atom alkanols), orpolyhydric alcohols such as glycols, glycerine, diethylene glycol,pentaerythritol, sorbitol, mannitol, ethyl alcohol, butyl alcohol,octadecyl alcohol, etc., e.g. glyceryl stearate, glyceryl palmitate,glycol distearate, glycol dilaurate, glycol diacetate, monoacetin,triacetin, glyceryl oleate, ethylene glycol stearate; such esters ofpolyvalent alcohols can in a given case be etherified, benzyl benzoate,dioxolane, glycerine formal, tetrahydrofurfuryl alcohol, polyglycolethers with 1 to 12 carbon atom alcohols, dimethyl acetamide, lactamide,lactates, e.g., ethyl lactate, ethyl carbonate, silicones (especiallymiddle viscosity dimethyl polysiloxane).

Other adjuvants can also be substances which bring about decomposition(so-called explosives) such as: cross-linked polyvinyl pyrrolidone,sodium carboxy methyl starch, sodium carboxy methyl cellulose ormicrocrystalline cellulose. Likewise, known coating agents such as e.g.polyacrylates, cellulose ethers and the like can be used.

For the production of solutions, there can be used water ofphysiologically compatible organic solvents, as for example, ethanol,1,2-propylene glycol, polyglycols, e.g., diethylene glycol, triethyleneglycol and dipropylene glycol and their derivatives dimethyl sulfoxide,fatty alcohols, e.g., stearyl alcohol, cetyl alcohol, lauryl alcohol andoleyl alcohol, triglycerides, e.g. glyceryl olelate glyceryl stearate,glyceryl palmitate, and glyceryl acetate, partial esters of glycerine,e.g., glyceryl monostearate, glyceryl distearate, glycerylmonopalmitate, paraffins, and the like.

For injectable solutions or suspensions, non-toxic parenterallycompatible diluting agents or solvents can be used, for example: Water,1,3 butane diol, ethanol, 1,2-propylene glycol, polyglycols in a mixturewith water, Ringer's solution, isotonic solution of sodium chloride oralso hardened oils including synthetic mono or diglycerides or fattyacids like oleic acid.

Known and customary solution assistants or emulsifiers can be used inthe production of the preparations. The following are examples ofsolution assistants and emulsifiers which can be used:Polyvinylpyrrolidone, sorbitan fatty acid esters such as sorbiantrioleate, phosphatides such as lecithin, acacia, tragacath,polyoxethylated sorbitan monooleate and other ethoxyated fatty acidesters of sorbitan, polyoxyethylated fats, polyoxyethylatedoleotriglycerides, linolized oleotriglycerides, polyethylene oxidecondensation products of fatty alcohols, alkyl phenolene or fatty acidsor also 1-methyl-3-(2-hydroxyethyl)imidazolidone-(2). The termpolyoxyethylated means in this context that the substances in questioncontain polyoxyethylene chains whose polymerization is generally between2 to 40 and especially between 10 to 20.

Such polyoxyethylated substances can be obtained, for example, byreacting compounds containing hydroxyl groups (e.g. mono or diglyceridesor unsaturated compounds such as, e.g., those containing the oleic acidresidues) with ethylene oxide (e.g. 40 moles ethylene oxide per moleglyceride). Examples of oleotriglycerides are olive oil, peanut oil,castor oil, sesame oil, cotton seed oil and corn oil. [See also Fiedler,Lexicon der Hilfastoffe fur Pharmazie, Kosmetik and angrezende Gebiete[Lexicon of Adjuvants for Pharmacy, Cosmetics an Related Areas] pp.191–195 (1971)].

Furthermore, there can be added preservatives stabilizers, buffers, forexample, calcium hydrogen phosphate, colloidal aluminum hydroxide, tastecorrectives, antioxidants and complex formers (for example, ethylenediamine tetraacetic acid) and the like. In a given case forstabilization of the active molecule, the pH is adjusted to about 3 to 7with physiologically compatible acids or buffers. Generally, there ispreferred as neutral as possible to weak acid (to pH 5) pH value.

As antioxidants, there can be used, for example, sodiummeta bisulfite,ascorbic acid, gallic acid, alkyl gallates, e.g., methyl gallate andethyl gallate, butyl hydroxyanisole, nordihydroguararetic acid,tocopherols as well as tocopherol and synergists (materials which bindheavy metals by complex formation, for example, lecithin, ascorbic acid,phosphoric acid). The addition of synergists increases considerably theantioxidant activity of tocopherol. As preservatives, there can be used,for example, sorbic acid, p-hydroxybenzoic acid esters (for example,lower alkyl esters such as the methyl ester and the ethyl ester) benzoicacid, sodium benzoate, trichloroisobutyl alcohol, phenol, cresol,benzethonium chloride, and formalin derivatives.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the following examples, the following abbreviations apply: IPF,inhibitory protein factor; BSA, bovine serum albumin; EDTA,(ethylenedinitrilo)-tetraacetic acid; GABA, γ-aminobutyric acid; HEPES,4-(2 hydroxyethyl)-1-piperazine-ethanesulfonic acid; PAE,polyethyleneimine; PMSF, phenylmethyl sulfonyl fluoride; SDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electrophoresis; V-typeH⁺-ATPase, vacuolar-type proton translocating ATPase.

In the following examples, the materials utilized were obtained asfollows: Polyethyleneimine (PAE)-1000 anion-exchanger was purchased fromAmicon Corporation. Hydroxylapatite HPT was from Bio-Rad. Superdex S20026/60 and Mono Q 5/5 columns were purchased from Pharmacia.[³H]Glutamate (50 Ci/mmol) was purchased from Amersham Corporation.CytoScint ES scintillation fluid was from ICN. Whatman GFC filters werepurchased from VWR. Ammonium sulfate was from Mallinckrodt. Protein wasquantified with the Coomassie Protein Assay Kit from Pierce, usingbovine serum albumin as standard. All other chemicals and chromatographymedia were purchased from Sigma.

Example 1 Purification and Use of IPF

In this example, the purification and use of an inhibitor of synapticglutamate uptake is described. The example is divided into threesections: A) preparation of the synaptic vesicles and synaptosomes, B)purification of inhibitory protein factor (IPF), and C) inhibition ofglutamate uptake by synaptic vesicles with IPF. All of the followingprocedures were performed on ice or in a 4° C. cold-room unlessotherwise noted.

A. Preparation of Synaptic Vesicles and Synaptosomes

Bovine synaptic vesicles were prepared by a slight modification of theprocedure described by Tabb et al. [(1992) J. Biol. Chem. 267,15412–15418]. Briefly, bovine brains were obtained from the localslaughterhouse. On ice, the meninges were removed form each brain, thecerebral cortex dissected, and excess white matter removed. About 300grams of cortex were briefly blended in 600 ml of 0.32 M sucrose, 1 mMNaHCO₃, 1 mM magnesium acetate, 0.5 mM calcium acetate, 0.2 mMphenylmethylsulfonyl fluoride (solution A) in a Waring blender with 3×7second bursts; solution A was used in all steps unless noted otherwise.

The homogenate was diluted to two liters in solution A and wasrehomogenized in a 300-ml tight fitting Teflon-glass homogenizer(Kontes, Vineland, N.J.) with two strokes, at 1900 rpm. Each batch wasdiluted to 3 liters and was centrifuged in a Sorvall GSA rotor at 2500rpm for 10 minutes. The supernatant (S1) was saved, and the pellets (P1)from various batches were pooled, diluted to 4 liters, rehomogenized andrecentrifuged at 2500 rpm for ten minutes. This supernatant (S1) waspooled with the previous S1 and was centrifuged in a GSA rotor at 13,000rpm for 15 minutes. This pellet was saved, and the supernatant wasdiscarded.

The P2 was resuspended in solution A to 1.6 liters. This suspension wasdiluted with an equal volume of 1.28 M sucrose, to make the finalconcentration of sucrose 0.8 M, and was then centrifuged at 13,000 rpmin a GSA rotor for 45 minutes. The floating myelin bands and supernatantwere aspirated, and the pellets (synaptosomes) were saved. Thesynaptosomes were resuspended in two liters of ice-cold lysing buffer (6mM Tris-maleate, pH 8.1), diluted to 8 liters lysing buffer,mechanically stirred at 4° C. for 45 minutes and were then centrifugedat 19,000 rpm in a Sorvall SS-34 rotor for 15 minutes. The supernatantwas then concentrated from 8 liters to 800 ml in an Amicon spiralultraconcentrator, equipped with an S1Y30 cartridge (30,000 molecularweight cutoff). The retentate was then centrifuged at 43,000 rpm in aBeckman 45Ti rotor ultracentrifuge rotor for 70 minutes.

The pellets (crude synaptic vesicles) were resuspended in 20 ml oflysing buffer and were layered over six discontinuous sucrose gradients(12 ml 0.4 M, 6 ml 0.6 M), and were then centrifuged at 35,000 rpm in aBeckman Type 45Ti rotor for two hours. The lysing buffer and 0.4 Msucrose layers (but not the 0.4 M, 0.6 M sucrose interface; it containsplasma membrane contaminants) were removed, diluted with lysing bufferand were centrifuged at 47,000 rpm in a Beckman Ti50 rotor for 60minutes. The pellets were saved and were either resuspended in solutionB (0.32 M sucrose, 1 mM NaHCO₃, 1 mM dithiothreitol) at about 5 mg/mland stored in liquid nitrogen and were stable for at least one month.

Bovine synaptosomes used in FIG. 5 (see description below) werecollected during the preparation of synaptic vesicles [Tabb et al.(1992) J. Biol. Chem. 267, 15412–15418]. Synaptosomes were resuspendedin normal Krebs-Ringers (0.15 M NaCl, 6.2 mM KCl, 1.2 mM Na₂HPO₄, 1.2 mMMgSO₄, 10 mM glucose, 20 mM Tris-HEPES, pH 7.4) or low Na⁺ Krebs-Ringers(substituting 0.15 M choline chloride for NaCl) prior to assay.

B. Purification of IPF

25 calf brains were obtained fresh from a local slaughterhouse, and thecerebellum, brain stem, and excess white matter removed to yield5,500–6,000 grams of cerebral tissue. A Waring blender was used to mince300 grams of cortex at a time in 800 ml of 1 mM PMSF, 1 mM EDTA, 5 mM2-mercaptoethanol, 6 mM Tris-HCl, pH 8.3 (lysing buffer). The blendedmaterial was then diluted to ˜40 liters in lysing buffer and homogenizedto smoothness by passing through a large, continuous-flow homogenizer.The entire suspension was then centrifuged at 13,000 rpm (27,300g_(max)) for 15 min in a Sorvall GSA rotor. The resulting supernatant(˜25 liters) was concentrated to 10 liters in an Amicon spiralultra-concentrator equipped with a S1Y30 cartridge (30,000 molecularweight cut-off) prior to further fractionation.

The inhibitory protein factor was purified to apparent homogeneity asfollows:

Step 1: Ammonium sulfate precipitation—The crude cellular extract (˜10liters) was adjusted to 45% saturation with ammonium sulfate andincubated for 30 min. The precipitate was collected by centrifugationfor 15 min at 13,000 rpm (27,300 g_(max)), resuspended to 2.5 liters inlysing buffer and dialyzed overnight against 55 liters of same. Thedialyzed sample was then clarified by centrifuging for 70 min at 45,000rpm (235,400 g_(max)) in a Beckman Type 45Ti rotor. The resultingsupernatant (2.6 liters, 24,000 mg protein) was generally storedovernight at −20° C. prior to anion-exchange chromatography.

Step 2: Anion-exchange chromatography—One half of the 45% ammoniumsulfate precipitate at a time (˜1.3 liters) was loaded onto a PAE-1000column (7.5 cm×32 cm) equilibrated with lysing buffer, at a flow-rate of50 ml/min. After collection of the flow-through fraction, bound proteinwas eluted with 2 liters each of 0.2, 0.5, and 1.0 M NaCl dissolved inthe column buffer. The 0.5 M NaCl eluate (1.5 liters) was dialyzedovernight against 58 liters of a solution containing 1 mM MgCl₂, 0.2 mMPMSF, and 10 mM Tris-maleate, pH 8.0 (HAP column buffer). The 0.5 M NaCleluate was usually fractionated on hydroxylapatite immediately followingdialysis.

Step 3: Hydroxylapatite column chromatography—This step was typicallyrun twice, with each run utilizing one of the two 0.5 M NaCl eluatesobtained from the PAE-1000 column. The dialyzed PAE 0.5 M eluate (1.6liters, 3,500 mg protein) was applied to a hydroxylapatite column (7.5cm×9 cm) equilibrated with HAP column buffer at a flow-rate of 20ml/min. Bound protein was eluted with increasing steps of potassiumphosphate (0.01, 0.05, 0.1, and 1 M) dissolved in HAP column buffer. The0.05 M eluate was collected and later combined with the same fractionobtained from the second column run. The combined HAP 0.05 M eluateswere adjusted to 80% saturation with ammonium sulfate, and theprecipitates collected and resuspended to 200 ml in a solutioncontaining 1 mM EDTA, 0.2 mM PMSF, and 10 mM Tris-maleate, pH 7.0(yellow column buffer). This was dialyzed overnight against two 18-literchanges of the same.

Step 4: Reactive Yellow-86 chromatography—The dialyzed 0.05 M phosphateeluate from the hydroxylapatite column (250 ml, 1,800 mg protein) wasloaded onto a Reactive Yellow-86 agarose column (4.5 cm×22 cm)equilibrated with yellow column buffer. Approximately 95% of loadedprotein does not bind to the column and is collected in theflow-through. Bound protein is eluted with successive steps of 0.06,0.3, and 1 M NaCl dissolved in column buffer, at a flow-rate of 14ml/min. The 0.3 M NaCl eluate (250 ml, 60 mg protein) was adjusted to80% saturation with ammonium sulfate and the precipitate collected aspreviously described. The precipitate was resuspended to 10 ml in yellowcolumn buffer and dialyzed overnight against two 5-liter changes of thesame.

Step 5: Gel filtration on Superdex S200—The dialyzed Yellow-86 0.3 MNaCl eluate (10 ml, 18–20 mg protein) was applied to a Superdex S-20026/60 column (62 cm×65 cm) equilibrated with a solution containing 75 mMKCl, 1 mM EDTA, 0.2 mM PMSF, and 10 mM Tris-maleate, pH 7.0. The columnwas run at 1 ml/min, and 6 ml fractions collected (60 total). Typically,fractions 23–30 were pooled, and an 80% ammonium sulfate precipitatecollected as previously described. Precipitate was resuspended in 2 mlof a solution containing 1 mM EDTA, 0.2 mM PMSF, and 10 mM Tris-maleate,pH 7.0, and dialyzed against 4 liters of the same for 4 hours just priorto sucrose gradient centrifugation.

The estimated Stokes radius was determined by utilizing plots of(—logK_(av))^(1/2) vs. the Stokes radius of standard proteins (ferritin,79 Å; BSA, 35 Å; and myoglobin, 17 Å) according to [Siegel and Monty(1966) Biochim. Biophys. Acta 112, 346–362].

Step 6: Sucrose density gradient centrifugation—The dialyzed, ammoniumsulfate precipitate from the Superdex S-200 column (2.5 ml, 20–25 mgprotein) was layered onto two 36 ml 5–20% sucrose gradients developed ina solution containing 50 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, and 10 mMHEPES, pH 7.4. Gradients were centrifuged for 43 hr at 28,000 rpm(140,000×g_(max)) in a Beckman SW28 rotor. A gradient containingcatalase (11.3 S), BSA (4.3 S), and myoglobin (2.1 S) was also run inparallel in order to determine the sedimentation coefficients in TableII below [Martin and Ames (1960) J. Biol. Chem. 236, 1372–1379].Typically, thirty-six 1-ml fractions were collected from each gradientby puncturing the tube bottoms.

Step 7: HPLC anion-exchange chromatography—Peak inhibitory fractionsfrom the sucrose gradients (typically fractions 22–26, ˜2 mg protein)were pooled and applied to a Pharmacia Mono Q 5/5 anion-exchange columnequilibrated with a solution containing 75 mM NaCl, 1 mM EDTA, 0.2 mMPMSF, and 20 mM Tris-HCl, pH 7.6. Bound protein was eluted with a linearNaCl gradient (75–538 mM) developed over 60 min at a flow-rate of 1ml/min. Fractions were collected (60×1 ml), and 10-μl aliquots wereassayed for inhibitory activity. Controls were performed by assayingaliquots from an identical gradient run in the absence of added protein.

Typical results of the procedure used to purify IPF α and IPF β areshown in Table I and in FIGS. 2 and 3 (described below). Hydroxylapatitechromatography at pH 8.0 in the presence of Mg²⁺ proved to be a criticalstep during purification. Under these conditions, about 50% of theinhibitory activity was found in the 0.05 M phosphate eluate andresolved from a second peak of inhibitory activity present in the 0.1 Meluate. This accounted for the 50% loss of total inhibitory activityseen at this step. The activity in 0.1 M eluate correlated with a 73 kDaprotein which may represent a proteolytic digestion product of IPF (EDÖand TU, unpublished observation).

The dye Reactive Yellow 86 was found to have a particularly selectiveaffinity for IPF. The 0.3 M NaCl eluate from this column provided thefirst glimpse of the IPF αβγ triplet against the protein background(FIG. 2, lane 6, described below). Greater than 90% of the loadedprotein did not bind to this column and, although the column effluentcontained the majority of the inhibitory activity, no increase inspecific activity was achieved.

Sucrose gradient centrifugation proved effective in the purification ofIPF because of the anomalous sedimentation behavior of the IPF triplet.IPF α,β, and γ all migrated identically in 5–20% sucrose gradients, withan apparent sedimentation coefficient of 4.3 S. This step usuallyyielded at least one fraction that was essentially a purifiedpreparation of IPF αβγ.

These steps and their resulting levels of purification are summarized inTable I, indicating a 1,160-fold to 1,280-fold purification for IPF αand IPF β respectively.

TABLE I Purification of IPF from calf brain cytosol. Total TotalSpecific Protein IC₅₀ Activity Yield Activity Purificat. Fraction (mg)(mg/.12 ml) (U) (%) (U/mg) (−fold) Cytosol 69,960 0.50 139,920 100 2 145% AS 26,920 0.25 107,680 77 4 2 PAE 6,952 0.081 85,827 61 12 6 0.5MHAP 1,824 0.040 45,600 33 25 13 0.05M Yellow 58 0.011 5,273 3.8 91 460.3M Superdex 23 0.0050 4,600 3.3 200 100 Peak Sucrose 2.5 0.0011 2,2731.6 909 455 Peak Mono Q 0.25 0.00043 581 0.42 2,326 1,160 IPF α Mono Q0.25 0.00039 641 0.46 2,564 1,280 IPF β

FIG. 2 displays SDS-PAGE profiles of the products in the steps of thepurification procedure. The starting material and fractions containingthe peak inhibitory activity from the various purification steps weredissociated by boiling for 2 min in the presence of 1% SDS, 5%2-mercaptoethanol, 10% glycerol, and 63 mM Tris-HCl, pH 6.8, andsubjected to electrophoresis on a 7.5% SDS-polyacrylamide gel. Gelstaining was with Coomassie Brilliant Blue. Lane 1, 40 μg calf-brainhomogenate; lane 2, 40 μg crude cytosol; lane 3, 40 μg 45% ammoniumsulfate precipitate; lane 4, 40 μg PAE 0.5 M NaCl eluate; lane 5, 40 μgHAP 0.05 M phosphate eluate; lane 6, 40 μg Yellow 86 0.3 M NaCl eluate;lane 7, 20 μg gel filtration peak; lane 8, 5 μg sucrose gradient peak;lane 9, 1.5 μg Mono Q-purified IPF α; lane 10, 1.5 μg Mono Q-purifiedIPF β.

FIG. 3 compares the SDS-PAGE profile, (upper panel), of peak inhibitoryfractions as eluted from Mono Q HPLC, (lower panel). Peak inhibitoryfractions from two 5–20% sucrose density gradients (12 ml, 2 mg protein)were applied to a Mono Q 5/5 anion-exchange column equilibrated in 75 mMNaCl, 1 mM EDTA, 0.2 mM PMSF, 20 mM Tris-HCl, pH 7.6. Bound protein waseluted in sixty 1-ml fractions by a linear NaCl gradient (75–538 mM)developed over 60 min. Aliquots of 10 μl were assayed for inhibitoryactivity (upper panel) and for composition by SDS-PAGE (lower panel).All of the inhibitory activity eluted between gradient fractions 24 and35. Samples for SDS-PAGE were dissociated and electrophoresed. Gelstaining was with Coomassie Brilliant Blue.

FIG. 3 shows that high resolution anion-exchange chromatography canpartially resolve IPF αβγ into individual components based on assumeddifferences in net negative charge. It can also be concluded from thisfigure that both IPF α and IPF β possess inhibitory activity. Fraction30 (see FIG. 2) usually contained a mixture of IPF α,β and γ,reminiscent of the starting material, while fraction 29 usuallycontained roughly equal amounts of IPF β and γ. Fraction 28 containedvery pure IPF β (135 kDa), and later fractions contained pure IPF α (138kD).

C. Inhibition of Glutamate Uptake By Synaptic Vesicles

The uptake of [³H]-glutamate into synaptic vesicles was assayed using amodification of the filtration procedure described in [Kish and Ueda(1989) Meth. Enzymol. 174, 9–25]. Briefly, synaptic vesicles (30–50 μgprotein) were suspended in 120 μl of an incubation medium consisting of0.23 M sucrose, 4 mM KCl, 4 mM MgSO₄, 2 mM aspartate, 10 mM methioninesulfoximine, 1 mM spermine, ±2 mM ATP, ±IPF sample 10 mM HEPES, pH 7.4,and 50 μM [³H]glutamate (specific activity, 0.017 Ci/mmol). Glutamateuptake was initiated by transferring the mixtures from ice to a 30° C.water bath, and the uptake reaction was allowed to proceed for 10 min.Baseline ATP-dependent glutamate uptake activity was calculated as theglutamate taken up in the presence of ATP minus that taken up in theabsence of ATP. Throughout this work, glutamate uptake activity refersto the ATP-dependent portion, which was typically greater than 90% ofthe total. One unit (U) of the inhibitory protein factor was defined asthe amount of protein required to inhibit 50% of ATP-dependent glutamateuptake over a period of 10 min at 30° C.

FIG. 4 shows that IPF α is a potent inhibitor of ATP-dependent glutamateuptake in synaptic vesicles. Purified bovine synaptic vesicles (50 μg ofprotein) were suspended in the glutamate uptake assay medium in thepresence of varying amounts of fraction 31 from the Mono Q column (0,0.1, 0.2, 0.3, 0.4, 0.5, 0.65, 0.76, 0.86, 0.97, 1.1, 1.3, and 1.6 μgprotein). Using a molecular weight of 138,000 for IPF α, these amountswere converted to 26 nmol IPF α/liter. Uptake was allowed to proceed for10 min at 30° C. Values for % of control were calculated relative to theATP-dependent uptake in samples containing equivalent amounts offraction 31 from a Mono Q gradient run in the absence of loaded protein.

A similar dose-response curve for IPF β was also generated and indicatedan IC₅₀ of 24 nM (data not shown). At approximately 100 nM, both IPF αand β inhibited ATP-dependent glutamate uptake by 90%. However, neitherIPF α nor IPF β had any effect on the ATP-independent component ofglutamate uptake even at concentrations up to 100 nM (data not shown).

In order to investigate the specificity of the inhibitory effectproduced by IPF, its effect on uptake in two other well characterizedsystems was examined: the Na⁺-dependent glutamate uptake system in thesynaptosomal plasma membrane and the ATP-dependent, reserpine-sensitivecatecholamine uptake system in chromaffin vesicles from the adrenalmedulla.

FIG. 5 depicts the results of this examination. Bovine synaptic vesicles(50 μg of protein) were suspended in glutamate uptake assay medium.Bovine synaptosomes (35 μg protein) were suspended in 0.12 ml ofKrebs-Ringer solution containing 1 mM spermine and 1 μM [³H]glutamate(1.67 Ci/mmol) in the presence of Na⁺ (150 mM) or choline (150 mM).Bovine chromaffin vesicles (45 μg protein) were suspended in 0.12 ml ofa solution containing 0.3 M sucrose, 1 mM spermine, 10 mM MgSO₄, 5 mMATP, 10 mM HEPES, pH 7.0 and 50 μM [³H]norepinephrine (0.017 Ci/mmol)with or without 1 μM reserpine. Each membrane mixture also containedeither 0, 26, or 100 nM IPF α. Glutamate uptake was allowed to proceedfor 10 min at 30° C. and 30 min at 37° C. for norepinephrine uptake.Reaction was terminated by filtration as described in Methods. Valuesfor % of control were calculated relative to the specific uptakeactivity obtained in the absence of IPF α. Uptake into synaptic vesiclesis represented by ATP-dependent uptake, that into synaptosomes byNa⁺-dependent uptake, and that into chromaffin vesicles byreserpine-sensitive uptake.

Results in FIG. 5 indicate that IPF a exhibited no inhibitory effect onNa⁺-dependent glutamate uptake into bovine synaptosomes. Moreover, IPF αhad only a minimal effect (˜18% inhibition) on norepinephrine uptakeinto bovine chromaffin vesicle ghosts at 100 nM, a concentration thatinhibited vesicular glutamate uptake by 90%.

The precise mechanism by which IPF leads to inhibition of vesicularglutamate uptake remains to be determined. Since transport of glutamateinto synaptic vesicles is a coupled process, the possibilities forinhibiting such a system are multiplied by the number of potentialcoupling sites. Indirect modes of inhibition would include inhibitingthe activity of the V-type H⁺-ATPase, increasing the passivepermeability of the vesicle membrane to protons, or causing ageneralized increase in membrane permeability (a detergent-like effect).The results in FIG. 5 indicate that these possibilities are unlikely. IfIPF were inhibiting the action of the V-type H⁺-ATPase (either ATPhydrolysis or proton pumping), it would be expected that norepinephrinetransport into chromaffin vesicles would also be inhibited. Similarly,any inhibitor which has a generalized protonophore activity would alsolead to decreased uptake into chromaffin vesicles. FIG. 5 shows thatnorepinephrine transport is hardly affected by IPF concentrations up to100 nM. Finally, the generalized increase in membrane permeabilitycharacteristically caused by detergents and other amphiphilic moleculesshould have an effect on glutamate storage in synaptosomes as well as onnorepinephrine uptake. This was not observed.

Even though it has not been possible thus far to isolate purifiedGABAergic vesicles, preliminary experiments with mixed vesiclepreparations (containing both glutamate and GABA uptake activities) haveshown IPF to be just as potent an inhibitor of vesicular GABA uptake(data not shown).

Example 2 Characterization of IPF

Some of the physicochemical properties of IPF are summarized in TableII.

TABLE II Property IPF α IPF β IPF γ Relative 138,000 135,000 132,000Molecular Wt Molecular Wt 103,500 — — Stokes Radius 60 60 60 (angstroms)Sedimentation 4.3 4.3 4.3 Coefficient Partial Specific 0.717 — — volume(ml/gm) Frictional ratio 1.67 — — (f/f₀) Axial ratio 12 — — IC₅₀ (nM) 2624 —

IPF α, β and γ share highly similar physicochemical properties. Therelative apparent molecular weights determined by SDS-PAGE were 138,135, and 132×10³ for IPF α, β and γ, respectively. Their Stokes radiiand sedimentation coefficients were indistinguishable from each other,being 60 Å and 4.3S, respectively. The partial specific volume of IPF αwas calculated to be 0.717 ml/g from amino acid composition. Using thesevalues, an approximate molecular weight of IPF α in the native form wascalculated to be 103,000, according to the equation M=6 πηNas/(1-{umlautover (υ)}ρ), where N=Avogadro's number, η=viscosity of water at 20° C.,a=Stokes radius, s=sedimentation coefficient at 20° C., {umlaut over(υ)}=partial specific volume, and ρ=density of water at 20° C. [Siegeland Monty (1966) Biochim. Biophys. Acta 112, 346–362]. This molecularweight is significantly smaller than that determined by SDS-PAGE. Theexcessively high Stokes radius and low sedimentation coefficient valuesfor a globular protein indicate that IPF has an elongated shape. This isin agreement with the rather high axial ratio of 12 estimated from thecalculated frictional coefficient ratio of 1.67.

Calculations utilizing the Stokes radius, sedimentation coefficient, andpartial specific volume for IPF α indicate a native molecular mass of103 kDa. This is at odds with the apparent molecular weight of 138,000determined by SDS-PAGE. Additionally, the unexpectedly low sedimentationcoefficient of 4.3 S is not consistent with the large Stokes radius (60Å). These data collectively indicate that IPF α is a protein with amarkedly elongated structure. This hypothesis is further supported bythe large frictional coefficient and axial ratio (see Table II)determined for IPF α and by the relationship to α fodrin, itself alinear protein. How this rather eccentric structural characteristicmight contribute to the ability to inhibit glutamate uptake is notknown. The differences in charge between IPF α and IPF β, which renderthem separable by ion-exchange HPLC (see FIG. 3), apparently have littleeffect on inhibitory activity, as the two proteins share similar IC₅₀values.

FIG. 6A (SEQ ID NO:5) gives the results of partial sequencing of IPF α.N-terminal sequencing revealed that amino acids 1–20 of IPF α,β and γare identical with amino acids 26–45 of human α fodrin. Four furtherpeptides (amino acids 393–415, 621–636, 965–974, and 1086–1095)generated by partial digestion of IPF α confirmed the relationship to αfodrin. Amino acid sequences determined for IPF α are shown in boldfacein FIG. 6A within the initial 1200 amino acid residues of human α fodrinas determined by Moon and McMahon [(1990) J. Biol. Chem. 265,4427–4433]. The 20-mer beginning with Tyr²⁶ represents the N-terminus ofIPF α. The four internal sequences were determined by sequencingpeptides produced by proteolytic digestion of IPF α. The highlightedbond between Tyr¹¹⁷⁶ and Gly¹¹⁷⁷ represents the cleavage site forcalpain [Harris et al. (1988) J. Biol. Chem. 263, 15754–15761]. FIG. 6B(SEQ ID NO:6) shows the predicted sequence of IPF based upon sequencingof the N-terminus and the cleavage site for calpain as the C-terminus.Based upon this sequence, IPF α is estimated to have a true molecularweight of about 133 kD.

Despite this relationship, fodrin purified from whole-brain, accordingto the method described by Cheney et al. [(1986) Meth. Enzymol. 134,42–54], had no effect on glutamate uptake at concentrations up to 1 μM(data not shown).

Example 3 Treatment of IPF with Trypsin

IPF partially isolated as described in Example 1 was further treated byincubation at 30° C. for five minutes in the presence of trypsin (0.1μg). FIG. 7 shows the effect of trypsin exposure on IPF. Lane A showsthe SDS-PAGE profile of the partially purified IPF composition withouttrypsin treatment. Lane B shows the SDS-PAGE profile of the partiallypurified IPF composition with trypsin treatment. It is clear that the138 kD IPF band is not present, demonstrating the digestion of IPF bytrypsin. Lane C shows the SDS-PAGE profile of the partially purified IPFcomposition obtained in the presence of trypsin and pancreatic trypsininhibitor. Thus, it is clear that treatment of IPF with trypsin inducesdigestion of the IPF proteins.

The impact of trypsin digestion on glutamate uptake inhibition insynaptic vesicles was then determined. FIG. 8 depicts these results.Lane A depicts control glutamate uptake without additives. Lane Bdepicts glutamate uptake by synaptic vesicles in the presence of trypsinand pancreatic trypsin inhibitor. Lane C depicts glutamate uptake bysynaptic vesicles in the presence of partially purified IPF. Lane Ddepicts glutamate uptake by synaptic vesicles in the presence ofpartially purified IPF and trypsin. Lane E depicts glutamate uptake bysynaptic vesicles in the presence of partially purified IPF, trypsin andpancreatic trypsin inhibitor. While Lanes A and B show no significantglutamate uptake inhibition, both the unfragmented IPF in Lanes C and Eand the trypsin fragmented IPF of Lane D showed glutamate uptakeinhibition. Thus, it is clear that one or more products resulting fromtrypsin digestion of IPF inhibit glutamate uptake by synaptic vesicles.

Example 4 Animal Model for In Vivo Drug Screening

As described above, the present invention contemplates the use ofsynaptic vesicle glutamate uptake inhibitors for the treatment ofneurosynaptic disorders. Screening of glutamate uptake inhibitors for invivo-efficacy can be performed in the context of animal models. Oneanimal model is the Epileptic (EL) mouse.

The seizures in EL mice are inherited as a multifactorial trait and areconsidered model for human complex partial seizures with secondarygeneralization [Rise et al. (1991) Science 253 669–73]. The seizures inEL mice occur spontaneously or can be induced by rhythmic vestibularstimulation [Kurokawa et al. (1966) Prog. Brain Res. 21A 112–30]. Theseizures in EL mice generally begin at 80 to 100 days of age and arethought to originate in or near the parietal lobe and then spreadquickly to the hippocampus and to other brain regions [Seyfried (1992)Neurosciences 18 (Suppl. 2) 9–20].

In this example, it is shown that synaptic vesicle glutamate uptake isincreased in a brain region-specific manner in epileptic (EL) mice ascompared to age matched non-epileptic control mice. This increase wasobserved at an age when EL mice express handling-induced seizures, butit was not seen in young EL mice prior to seizure onset. The findingscould reflect an increase in glutamatergic synaptic vesicle number, withor without a change in the number of nerve terminals, or alternativelyan increase in the number of nerve terminals without an increase invesicles per terminal. A combination of these mechanisms might also beinvolved. It is also possible that this increase is due to alterationsin the synaptic vesicle such as regulation of transport by chloride oran internal volume change in the synaptic vesicle.

Synaptic vesicles isolated from various brain regions in EL mice priorto (46 days old) and after (approx. 400 days old) the onset of handlingor spontaneous seizures are studied. The EL mice were compared toage-matched nonepileptic control mice (DDY and APB).

Synaptic vesicles were prepared from four brain regions: cerebrum (minushippocampus), hippocampus, cerebellum and brain stem. The cerebrumtherefore contains the entire cerebral cortex, except for thehippocampus. Each preparation represents the synaptic vesicles from onemouse with the exception of the hippocampus, where each preparationrepresents 1–2 mice. Each tissue preparation represents different mice.

In brief, brain tissue was homogenized in solution A (0.32 M sucrose,0.5 mM calcium acetate, 1 mM magnesium acetate and 1 mM NaHCO₃). Thehomogenate was centrifuged at 10,000 rpm in a Sorvall SM-24 rotor for 20minutes. The pellets were lysed for 45 minutes in 6 mM Tris-maleate (pH8.1) and centrifuged at 19,000 rpm in a Sorvall SM-24 rotor for 15minutes. The supernatant was centrifuged at 43,000 rpm in a Beckman Ti45rotor for 70 minutes, and the crude synaptic vesicles were resuspendedin solution B (0.32 M sucrose, 1 mM dithiothreitol and 1 mM NaHCO₃).Synaptic vesicles were stored in liquid nitrogen until use, typicallywithin one week. Synaptic vesicles prepared in this manner exhibitedproperties essentially indistinguishable from those observed with highlypurified preparations.

Synaptic vesicle uptake activity was determined by the method describedabove. In the old mice, glutamate uptake activity was significantlyincreased in the EL mice cerebellum (minus hippocampus) compared to thecontrol DDY mice. No difference was observed between EL and control DDYmice in the other brain areas. In younger mice, no significantdifference in ATP-dependent glutamate uptake activity was observedeither in the cerebrum (minus hippocampus) or in the brain stem. Theseresults are set forth in Table III.

TABLE III Vesicular Uptake in EL and Control Non-Epileptic Mice Hippo-Cerebrum camp Cerebellum Brain Stem (pmol/mg (pmol/mg (pmol/mg (pmol/mgStrain Days Old protein) protein) protein) protein) EL 411 ± 10 1230 ±59  766 ± 107 358 ± 55 466 ± 66 DDY 395 ± 8  813 ± 69 689 ± 88  316 ± 11374 ± 43 ABP 424 ± 6  796 ± 64 632 ± 123 N.D. N.D. EL 46 ± 0 941 ± 61N.D. N.D.  872 ± 119 DDY 46 ± 0 906 ± 84 N.D. N.D. 1078 ± 181

The data suggest that ATP-dependent glutamate uptake increases inresponse to seizures. No difference were found between epileptic andnon-epileptic mice prior to the onset of seizures (46 days old). TheATP-dependent glutamate uptake was increased in the epileptic mice in abrain-region specific manner at an age when the EL mice had a longseizure history. The data therefore suggest that the increased synapticvesicle glutamate uptake in EL mice is not the initial cause of seizuredevelopment, but either an effect of the seizures or the cause ofcontinued seizures.

Previous studies of glutamate levels in EL mice were concerned withchanges in the total levels in brain tissue, including pools for bothmetabolism and synaptic release. It was difficult to draw coherentconclusions from some of these studies, as the results were not alwaysconsistent. Since the neurotransmitter pool represents a small portionof the total tissue glutamate content, these changes may not directlyreflect changes in the levels of glutamate to be released as aneurotransmitter. Recent findings suggest that enhanced aspartaterelease may be genetically associated with seizure susceptibility in ELmice.

While no differences were observed in other brain regions betweenepileptic and nonepileptic mice, it was observed that ATP-dependentglutamate uptake activity in the brain stem was higher in the 46 day oldmice compared to the 400 day old mice. It is likely that this relates tomaturation effects. It is unlikely that this relates to seizuredevelopment, as the control strain demonstrated uptake activityindistinguishable from that observed in the epileptic strain, regardlessof age.

Example 5 Polyclonal Antibodies to IPF

In this example, antibodies were raised that were demonstrated to bereactive with both intact fodrin and a fodrin fragment (i.e. IPF alpha).Moreover, following absorption of the antibody mixture on column ofimmobilized intact fodrin, antibodies reactive with intact fodrin wereremoved, leaving antibodies only reactive with the fodrin fragment.

Polyclonal antibodies to a decapeptide having a sequence correspondingto the N-terminal sequence of IPF α (anti-IPF α) were made in rabbits.The total protein from 50 ug Cytosol were then separated on SDS-PAGE.One strip of the gel was stained with Coomassie Blue. The proteins inthe remaining portion of the gel were transferred to nitrocellulose, thenitrocellulose was temporarily stained to visualize the lanes,destained, and blocked. The blot was cut into strips and each stripincubated with the appropriate primary antibody. After washing inbuffer, antibody binding was visualized using a goat anti-rabbit IgGconjugated to alkaline phosphatase from Bio-Rad.

The results of the immunoblotting are shown in FIG. 9. Panel A is a 7.5%polyacrylamide gel and containing 50 micrograms of cytosol. In this gel,the sample was probed with a 1:2000 dilution of unabsorbed rabbitanti-decapeptide. The gel shows that the unabsorbed antibody reacts withboth intact fodrin and IPF α.

The antibody used in Panel A was thereafter absorbed on a columncontaining purified fodrin coupled to sepharose 4B. Total protein from a100 ug Cytosol preparation was run (along with IPF alpha) on a gel (6.0%polyacrylamide gel) and transferred to nitrocellulose. The immunoblotwas probed with a 1:1000 dilution of antiserum. The results are shown inPanel B. The immunoblot shows binding of antibody to IPF α but noantibody binding to intact fodrin. This suggests that the conformationof intact fodrin is such that at least a portion of the regionrepresented by the decapeptide is sterically hidden.

Based upon the description and experimental materials presented above,it is clear that the present invention provides compositions and methodfor the treatment of neurosynaptic disorders in a subject.

1. A method for assessing overcoming synaptic vesicle glutamate uptakeinhibition activity, comprising a) providing: i) synaptic vesicles, ii)a composition comprising a purified fordin fragment having glutamateuptake inhibition activity, said fragment having an N-terminus and aC-terminus, wherein said N-terminus is Tyr26 of fodrin, and iii) acandidate compound; and b) combining said candidate compound with saidsynaptic vesicles and said fragment such that the effect of saidcandidate compound on glutamate uptake by said vesicle can be assessed.2. The method of claim 1, wherein said purified fragment comprises IPFα.
 3. The method of claim 1, wherein said purified fragment comprisesIPF β.
 4. The method of claim 1, wherein said purified fragmentcomprises IPF γ.
 5. A method for assessing overcoming synaptic vesicleglutamate uptake inhibition activity, comprising a) providing: i)synaptic vesicles, ii) a composition comprising a purified fragment ofIPF having synaptic vesicle glutamate uptake inhibition activity, saidfragment having an N-terminus and a C-terminus, wherein said N-terminusis Tyr26 of fodrin, and iii) a candidate compound; and b) combining saidcandidate compound with said synaptic vesicles and said fragment suchthat the effect of said candidate compound on glutamate uptake by saidvesicle can be assessed.
 6. The method of claim 5, wherein said fragmentcomprises a fragment of IPF α.
 7. The method of claim 5, wherein saidfragment comprises a fragment of IPF β.
 8. The method of claim 5,wherein said fragment comprises a fragment of IPF γ.
 9. The method ofclaim 1, wherein said purified fragment comprises a fragment of IPFα.10. The method of claim 5, wherein said purified fragment comprises afragment of IPFα.
 11. A method for assessing overcoming synaptic vesicleglutamate uptake inhibition activity, comprising: a) providing: i)synaptic vesicles, ii) a composition comprising a purified fragment offodrin having glutamate uptake inhibition activity, said fragment havingan N-terminus and a C-terminus, wherein said purified fragment comprisesa peptide having the amino acid sequence EAALTSEEVG within 150 aminoacids of the C-terminus of the peptide, and iii) a candidate compound;and b) combining said candidate compound with said synaptic vesicles andsaid fragment such that the effect of said candidate compound onglutamate uptake by said synaptic vesicles can be assessed.
 12. A methodfor assessing overcoming synaptic vesicle glutamate uptake inhibitionactivity, comprising: a) providing: i) synaptic vesicles, ii) acomposition comprising a purified peptide having glutamate uptakeinhibition activity with an N-terminus sequence comprising the aminoacid sequence YHRFK, and iii) a candidate compound; and b) combiningsaid candidate compound with said synaptic vesicles and said fragmentsuch that the effect of said candidate compound on glutamate uptake bysaid synaptic vesicles can be assessed.
 13. The method of claim 12,wherein said purified peptide has an N-terminus comprising the aminoacid sequence YHRFKELSTL (SEQ ID NO:3).
 14. The method of claim 13,wherein said purified peptide has an N-terminus comprising the aminoacid sequence YHRFKELSTLRRQKLEDSYR (SEQ ID NO:4).